Production and purification of monoclonal antibodies to Pisum and Avena phytochrome

At least nine monoclonal antibodies against phytochrome from Pisum sativum L. and 20 against phytochrome from Avena sativa L. have been obtained from mouse hybridomas that were produced by fusion of spleen cells with SP 2/O-Ag14 myeloma cells. Hybridomas were selected and cloned in a single step by plating on a semisolid methylcellulose medium. Eight antibodies to Pisum and one to Avena phytochrome were immunopurified from hybridoma medium or ascitic fluid. When necessary, secreted antibodies were verified to be against phytochrome by demonstrating to be against phytochrome by demonstrating immunoadsorption of phytochrome, detected as loss of photoactivity and-or by appearance of the approx. 120,000-dalton phytochrome band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation

Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-α–mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-α activity is regulated by Rack1–keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-α activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth.     

Use of biochemical markers to monitor changes in bone turnover in cynomolgus monkeys

The ovariectomized old cynomolgus monkey is a recognized model of human osteoporosis, and the same species can be used for the assessment of the efficacy and potential toxicity of agents intended to prevent or treat osteoporosis. Several assays have been developed that can measure the same biochemical markers of bone turnover as are used in human patients for the diagnosis and treatment follow-up of bone-related diseases, including osteoporosis. The aim of the present study was to describe the results obtained with these assays in normal control monkeys, their variations with age and sex, and their sensitivity in monitoring the bone turnover induced by ovariectomy in old skeletally mature cynomolgus monkeys. Seven old cynomolgus monkeys were bilaterally ovariectomized and 13 age-matched monkeys were sham-operated. Bone mineral density and biochemical markers were measured before and at regular intervals after surgery for up to 20 months. Total alkaline phosphatase (total ALP), bone-specific alkaline phosphatase isoenzyme (bone ALP) and osteocalcin (OC) were highly correlated to the decrease in bone mineral density (BMD) induced by ovariectomy. Deoxypyridinoline (DPD) measured by enzyme-linked immunoassay was insensitive to the bone resorption induced by ovariectomy, but cross-linked N-telopeptide (NTX-I) was higher in ovariectomized monkeys than in control monkeys. These results demonstrate that reliable biochemical parameters are available to adequately monitor and provide insight into osteoclastic bone resorption and osteoblastic bone formation, the two components of bone turnover in this animal model, and can thus be used to assess the efficacy and toxicity of potential therapeutic agents.     

Transfer of the Nonmatch-to-Goal rule in Monkeys across Cognitive Domains

To solve novel problems, it is advantageous to abstract relevant information from past experience to transfer on related problems. To study whether macaque monkeys were able to transfer an abstract rule across cognitive domains, we trained two monkeys on a nonmatch-to-goal (NMTG) task. In the object version of the task (O-NMTG), the monkeys were required to choose between two object-like stimuli, which differed either only in shape or in shape and color. For each choice, they were required to switch from their previously chosen object-goal to a different one. After they reached a performance level of over 90% correct on the O-NMTG task, the monkeys were tested for rule transfer on a spatial version of the task (S-NMTG). To receive a reward, the monkeys had to switch from their previously chosen location to a different one. In both the O-NMTG and S-NMTG tasks, there were four potential choices, presented in pairs from trial-to-trial. We found that both monkeys transferred successfully the NMTG rule within the first testing session, showing effective transfer of the learned rule between two cognitive domains.     

Audio-Visual Perception of 3D Cinematography: An fMRI Study Using Condition-Based and Computation-Based Analyses

The use of naturalistic stimuli to probe sensory functions in the human brain is gaining increasing interest. Previous imaging studies examined brain activity associated with the processing of cinematographic material using both standard “condition-based” designs, as well as “computational” methods based on the extraction of time-varying features of the stimuli (e.g. motion). Here, we exploited both approaches to investigate the neural correlates of complex visual and auditory spatial signals in cinematography. In the first experiment, the participants watched a piece of a commercial movie presented in four blocked conditions: 3D vision with surround sounds (3D-Surround), 3D with monaural sound (3D-Mono), 2D-Surround, and 2D-Mono. In the second experiment, they watched two different segments of the movie both presented continuously in 3D-Surround. The blocked presentation served for standard condition-based analyses, while all datasets were submitted to computation-based analyses. The latter assessed where activity co-varied with visual disparity signals and the complexity of auditory multi-sources signals. The blocked analyses associated 3D viewing with the activation of the dorsal and lateral occipital cortex and superior parietal lobule, while the surround sounds activated the superior and middle temporal gyri (S/MTG). The computation-based analyses revealed the effects of absolute disparity in dorsal occipital and posterior parietal cortices and of disparity gradients in the posterior middle temporal gyrus plus the inferior frontal gyrus. The complexity of the surround sounds was associated with activity in specific sub-regions of S/MTG, even after accounting for changes of sound intensity. These results demonstrate that the processing of naturalistic audio-visual signals entails an extensive set of visual and auditory areas, and that computation-based analyses can track the contribution of complex spatial aspects characterizing such life-like stimuli.     

Histone Chaperone NAP1 Mediates Sister Chromatid Resolution by Counteracting Protein Phosphatase 2A

Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. We found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. We conclude that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis.     

Retinal Vascular Caliber Is Associated with Cardiovascular Biomarkers of Oxidative Stress and Inflammation: The POLA Study

Purpose

Retinal vascular caliber has been linked with increased cardiovascular risk and is predictive of cardiovascular pathology, including stroke and coronary heart disease. Oxidative stress, as well as inflammatory mechanisms, plays a major role in the pathogenesis and progression of atherosclerosis, plaque rupture and vascular thrombotic propensity. The purpose of this study is to explore the relationship between retinal vascular calibers and biomarkers of oxidative stress and inflammation, in subjects free of cardiovascular pathology.

Patients and Methods

Cross-sectional analysis from a community-dwelling cohort comprising 1224 individuals aged 60 years and over, without a history of coronary or peripheral artery disease or stroke. Retinal vascular caliber was measured from fundus photographs using semi-automated standardized imaging software. Oxidative stress was evaluated using plasma superoxide dismutase 2 and glutathione peroxidase (GPx-3) activities, and inflammatory state was assessed using plasma high sensitivity C-reactive protein (hsCRP) and orosomucoid.

Results

In a multivariate model controlling for cardiovascular risk factors, larger retinal arteriolar caliber was independently related to higher level of GPx-3 activity (p = 0.003) whereas larger venular caliber was associated with higher levels of hsCRP (p = 0.0001) and orosomucoid (p = 0.01).

Conclusion

In the present study, biomarkers of oxidative stress regulation and inflammation were independently associated with retinal vascular calibers. This suggests that an assessment of retinal vessels may offer early and non-invasive detection of subclinical vascular pathology.     

Estimation of Parameters Influencing Waterborne Transmission of Infectious Hematopoietic Necrosis Virus (IHNV) in Atlantic Salmon (Salmo salar)

Understanding how pathogenic organisms spread in the environment is crucial for the management of disease, yet knowledge of propagule dispersal and transmission in aquatic environments is limited. We conducted empirical studies using the aquatic virus, infectious hematopoietic necrosis virus (IHNV), to quantify infectious dose, shedding capacity, and virus destruction rates in order to better understand the transmission of IHN virus among Atlantic salmon marine net-pen aquaculture. Transmission of virus and subsequent mortality in Atlantic salmon post-smolts was initiated with as low as 10 plaque forming units (pfu) ml⁻¹. Virus shedding from IHNV infected Atlantic salmon was detected before the onset of visible signs of disease with peak shed rates averaging 3.2×10⁷ pfu fish⁻¹ hour⁻¹ one to two days prior to mortality. Once shed into the marine environment, the abundance of free IHNV is modulated by sunlight (UV A and B) and the growth of natural biota present in the seawater. Virus decayed very slowly in sterilized seawater while rates as high as k =  4.37 d⁻¹ were observed in natural seawater. Decay rates were further accelerated when exposed to sunlight with virus infectivity reduced by six orders of magnitude within 3 hours of full sunlight exposure. Coupling the IHNV transmission parameter estimates determined here with physical water circulation models, will increase the understanding of IHNV dispersal and provide accurate geospatial predictions of risk for IHNV transmission from marine salmon sites.